Activation of NF-kB by DNA-damaging anticancer agents, including ionizing radiation (IR), has emerged as an important modulator of malignant cell behaviors, such as resistance to apoptotic cell death. This signal transduction pathway also serves as a paradigm to understand how nuclear DNA damage may induce nucleus-to-cytoplasmic signal transduction pathways. We have previously discovered a novel nucleus-to-cytoplasmic NF-kB signaling pathway induced by IR and other agents that can induce DNA double strand breaks (DSBs). This signaling pathway involves a post-translational modification (PTM) of NEMO (NF-kB essential modulator)/IKK3, the regulatory subunit of the I:B kinase (IKK) complex, by SUMO-1 (small ubiquitin-like modifier 1). We now generated a novel NemoDK knockin mice harboring a germ-line mutation of the sumoylation sites. In the current proposal, we will establish the physiological importance of this new NF-kB signaling pathway in response to IR by directly evaluating the role of NEMO sumoylation in vivo and further dissect the critical upstream and downstream mechanisms. The following three aims will address our central hypothesis that NEMO sumoylation plays a critical physiological role in mediating NF-kB activation by IR to modulate radiation sensitivity in vivo: Aim 1: Determine the roles of NEMO sumoylation in modulating radiation sensitivity in vivo. Aim 2: Reveal the upstream role of NEMO zinc finger in promoting NEMO sumoylation. Aim 3. Elucidate SUMO-1 specific downstream regulation of NEMO function. We believe that the proposed research is important for two major reasons. First, the described research will uncover novel NF-kB signal transduction mechanisms in response to DNA damage stimuli. Second, understanding the mechanisms of NF-kB activation by DNA damaging agents may help identify novel drug targets to improve the current methods of cancer therapy.